The AKT protein family, also known as protein kinases B (PKB), is known to be involved in a wide variety of biological processes including cell proliferation, differentiation, apoptosis, tumourigenesis, as well as glycogen synthesis and glucose uptake. These enzymes are members of the serine/threonine-specific protein kinase family.
The PKB/AKT pathway has been identified as an important regulator of cell survival signalling and apoptosis in cells. Signalling is thought to occur through a range of growth factor receptors including platelet derived growth factor, insulin growth factor and nerve growth factor, resulting in activation of phosphatidylinositol 3-OH kinase (PI-3K). This activation in turn leads to the generation of phosphatidylinositol(3,4,5)triphosphate (PIP3). Activated PIP3 binds to and in turn phosphorylates the enzyme PDK-1, the main activator of AKT, through its pleckstrin homology domain. Activated PDK-1 is responsible for a phosphorylation event at Thr308 of AKT, which induces a conformational change that facilitates further phosphorylation of AKT at Ser 473 by PDK-2.
PDK-1 phosphorylation of downstream kinases is not unique to AKT, as it has been reported to activate p70 S6 kinase and protein kinase C.
The activation of AKT influences multiple events within the cell including the inhibition of apoptosis, the progression of the cell cycle, cellular survival, metabolism, angiogenesis and hormone resistance.
Presently three family members of AKT have been identified, AKT 1, AKT 2 and AKT 3 (also known as PKBα, PKBβ and PKBγ). The family members share 80% amino acid sequence homology and all retain similar regional structure. They possess a C-terminal pleckstrin homology domain, a catalytic domain, a short q helical linker region and a carboxyl terminal domain. The PH domain permits binding of proteins to the cell membrane through a phospholipid interaction. The catalytic domain of AKT family members contains two residues essential for kinase activation, namely Thr308 and Ser 473. In turn AKT can phosphorylate any protein containing the RXRXXS/T-B motif where X represents any amino acid and B represents bulky hydrophobic residues.
Turning to the cellular function of AKT, hyperactivation of AKT has been linked to the inhibition of cellular apoptosis due to phosphorylation and negative regulation of the forkhead family of transcription factors which regulate various genes responsible for instigating death processes including FKHR, FKHRL1 and AFX. Conversely, AKT has been reported to up-regulate genes which are known to be anti-apoptotic including IKK and CREB. It is this mixture of positive and negative regulation which highlights the importance of AKT in regulating apoptosis. AKT promotes unwanted cell survival through its phosphorylation of several key apoptotic proteins including Bad and Pro-caspase 9, thus rendering them inactive and preventing signalling through this pathway. AKT activates and inhibits multiple mechanisms which have a major role in the progression of the cell cycle, ultimately leading to cell proliferation. The best characterised cell cycle regulator and tumour suppressor proteins p53 can be dysregulated via AKT phosphorylation and activation of the main p53 negative regulator MDM2. Phosphorylated MDM2 translocates to the nucleus where it prevents p53 transcription. The inhibition of p53 allows aberrant proliferation of the cell and progression towards a benign state.
In a similar fashion, AKT can also phosphorylate p27kip1 and p21; two main inhibitors of cycle progression, leading to loss of function, resulting in unchecked cell cycle progress and excessive proliferation.
AKT activation causes an increase in the rate of glycolysis by increasing the rate of glucose metabolism. It has also been reported that activated AKT stimulates the transport of amino acids and supports mTOR dependent increases in protein translation. Proangiogenic factors, such as vascular endothelial growth factor (VEGF), have been reported to activate AKT, ultimately resulting in inhibition of endothelial apoptosis, as well as activating endothelial nitric oxide synthase (eNOS). The sum result of this is rapid neovascularisation and cell migration.
Hypoxia driven angiogenesis, primarily mediated by hypoxia inducible factor (HIF 1α) can lead to the induction of multiple proteins including VEGF. Increased activated AKT has been reported to increase HIF-1α expression leading to an increase in angiogenesis independent of a hypoxic environment. Recent data has shown that HIF-1α activity in invasive breast cancer is correlated with increased activated AKT-1 phosphorylation.
Estrogen receptor (ER) and androgen receptor (AR) inhibitors, designed to inhibit cell signalling and induce apoptosis, are vital tools in cancer therapies. Incidence of resistance to these drugs arises rapidly in cancers including prostate, breast and ovarian. AKT has been reported to phosphorylate androgen receptors, leading to inhibition of AR activity and blockade of normal apoptotic signalling in prostate cancer induced by androgens.
In a similar manner, activation of AKT leads to phosphorylation of ERα resulting in an inhibition of tamoxifen mediated apoptosis or tumour regression, coupled with the creation of an estrogen independent signalling pathway. Activated AKT-2 has been identified as a promoter of ERα transcription in the presence or absence of estrogen increasing the rate of proliferation of breast cancer cells.
Hyperactivated AKT has been reported in a range of cancers compared to normal tissues including breast, lung, prostate, gastric, ovary, pancreas, thyroid, glioblastoma and haemological cancers. Phosphorylation of AKT has also been associated with clinical characteristics including increased stage and grade of tumour and increased poor prognosis. The activation of AKT can arise from a number of different genetic mutations in the AKT/PI-3K pathway.
Somatic mutations in the PI-3KCA gene have been widely reported in a large variety of tumours including breast, prostate and head and neck. A large number of these mutations will increase copy number of the gene leading to an increase in PI-3K activity. A recent study has identified a PI-3K mutation which selectively phosphorylates AKT in colon cancer which results in increase cell proliferation and invasion).
Any mutation which increases the activity of the PI-3K pathway will ultimately result in an increased activation of AKT. Gene amplifications are common occurrences in cancer. Amplifications of AKT-2 have been reported in ovarian, pancreatic, breast and head and neck squamous cell carcinoma. No amplifications or mutations in AKT-3 have been reported to date although deletion mutations leading to hyperactivation and amplification mutations have been reported associated with AKT-1. One mutation; E17K, results in pathological localization of AKT-1 to the cell membrane, inducing its activation and resulting in downstream signalling and cellular transformation. In vivo, this mutation has been shown to induce leukaemia in mice.
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumour suppressor gene known to negatively regulate AKT function. In cancer, loss of PTEN function results in constitutive phosphorylation of AKT and other downstream effectors of the PI-3K pathway. Loss of PTEN, due to deletion mutations or promoter methylation, has been reported in a number of different cancers including glioblastoma, endometrial, lung, breast, prostate and thyroid. This loss is commonly associated with hyperactivation of AKT. Recent studies have shown that loss of heterozygosity (LOH) at the PTEN gene was directly correlated to increased AKT activation and chemoresistance in gastric carcinomas and decreased progesterone receptor expression in breast carcinomas.
AKT activation is commonly initiated at the cell surface through a signalling event at a receptor, usually one of the tyrosine kinase family. Two tyrosine kinase receptors commonly amplified or overexpressed in cancer are HER2 and EGFR. In HER2 overexpressing tumours there is often a hyperactivation of AKT, this has been reported in ovarian, stomach and bladder cancer. Similarly in EGFR overexpressing tumours, particularly those with the EGFRvIII activating mutation, selective activation of AKT has been reported in a range of cancers including non-small cell lung cancers, breast, ovarian and most commonly high grade gliomas.
Examples of AKT inhibitors are provided in WO 2008/070134, WO 2008/070016 and WO 2008/070041. These documents provide specific naphthyridine compounds substituted with a five membered heterocycle.